Sensitive and Quantitative MS-bases Glycomic Mapping Platform

基于 MS 的灵敏定量糖组图谱平台

• 批准号: 10019565
• 负责人: Yehia Mechref
• 金额: $ 28.4万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10019565
• 关键词: Acids; Adhesions; Algorithms; Amino Acids; Area; Asparagine; Automation; Biological; Biological Assay; Biological Process; Biology; Carbon; Carbon nanoparticle; Cell Culture Techniques; Cell Differentiation process; Cells; Chromatography; Computer software; Cultured Cells; Cystic Fibrosis; Data; Data Set; Degenerative polyarthritis; Detection; Deuterium; Development; Diagnostic; Digestion; Disease; Drug Targeting; Energy Transfer; Enzymes; Evaluation; Exhibits; Funding; Future; Glycoproteins; Glycosides; Goals; Human; Immune response; Immune system; Isomerism; Isotope Labeling; Isotopes; Label; Laboratories; Light; Link; Lipids; Liquid Chromatography; Liquid substance; Malignant Neoplasms; Malignant neoplasm of esophagus; Malignant neoplasm of liver; Manuals; Mass Spectrum Analysis; Medical; Metabolic; Methods; Modeling; Monitor; Monosaccharides; Names; Natural graphite; Peptide N-glycohydrolase F; Phase; Physiological; Plasma; Polysaccharides; Preparation; Proteins; Proteomics; Public Health; Reaction; Recombinant Proteins; Reporting; Research; Research Personnel; Running; Sampling; Scientist; Serine; Signal Transduction; Signaling Protein; Site; Software Tools; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; System; Threonine; Tissue Sample; Tissues; Tyrosine; Variant; alpha-Fetoproteins; ammonium hydroxide; analytical method; analytical tool; base; cancer biomarkers; carbohydrate structure; experimental study; glycoproteomics; glycosylation; graphene; improved; informatics tool; interest; ionization; isotope incorporation; malignant breast neoplasm; methyl iodide; microwave electromagnetic radiation; open source; pathogen; preservation; rapid technique; side effect; software development; stable isotope; therapeutic protein; tool; tool development

Glycomics has emerged as an interesting yet challenging area of research in biology. Glycans function in numerous important biological areas such as, but not limited to: the immune system, cell development, cell differentiation/adhesion, host-pathogen interactions, protein signaling, and protein stabilization. Abnormal glycosylation has been associated with several diseases including cancer, cystic fibrosis, and osteoarthritis. Glycomics/glycoproteomics studies aim to quantify and characterize glycan structures (including linkage and positional isomers), protein attachment sites, and the protein’s identity. Approximately 50% of mammalian proteins are glycosylated but their abundance is rather low compared to non-glycosylated proteins. Furthermore, numerous glycans can occupy the same glycan attachment site on a protein; that is the same protein pool can have several different types of glycans attached to the same site, each with a potentially different function or a particular activity. Protein glycans are divided into two classes based on their amino acid attachment sites: asparagine for N-glycans and threonine, serine, and tyrosine for O-glycans. A strategy that has been successfully employed to investigate N-glycans in cells is to release or separate the glycans from proteins with the enzyme PNGase F, and study the global glycan composition of a sample. A drawback to this approach is that, so called, native glycans possess low ionization efficiencies which make their analysis by mass spectrometry quite difficult; however, this sensitivity issue can be overcome by permethylating glycans. Glycans have many isomers which can make their accurate analysis by LC-MS/MS difficult if the isomers cannot be resolved. This proposal demonstrates that we are able to separate permethylated glycan isomerss with a heated PGC column before mass spectrometry analysis (Aim 1), resulting in an extremely sensitive assay to accurately characterize and quantitate glycan isomers in biological samples. Although the separation of isomeric glycans has been previously reported, prior studies only resolved native and reducing end labeled glycan structures. Owing to the fact that permethylated glycans exhibit ionization efficiencies at least two orders of magnitude higher than the aforementioned structures, the importance of the increase in sensitivity, for the detection of structures at physiological concentrations, that accompanies isomeric separation of permethylated glycans (Aim 1) cannot be overstated. To overcome the variation in ionization efficiency between LC-MS samples, we have successfully permethylated glycans with various stable isotope combinations to achieve unprecedented quantitative glycan comparisons across samples derived from cell culture experiments, biological fluids, and biological tissues. Through the implementation of our multi-level isotopic labeling strategies (metabolic 15N labeling, 18O reducing end labeling and multiplex permethylation), the number of potential multiplexed samples can be increased from eight, the previous maximum, to 16 and 32 for biological fluid and tissue samples and cell culture samples, respectively (Aim 2). High throughput isomeric characterization glycans derived from biological samples can be attained by combining the methods described in Aims 1 and 2. While PNGase F is used extensively to release N-glycans from proteins, no such enzyme exists or has been discovered for O-glycans. We have developed a rapid method, RAIDR (Rapid Ammonium hydroxide Isobutyric acid O-glycan Deglycosylation Reaction), for selectively releasing O-glycans; RAIDR leaves the protein and N-glycans unscathed which allows for compatible downstream analyses (Aim 3). In addition to improving LC-MS analytical methods, we are also proposing the addition of graphene nanosheets and carbon nanoparticles to MALDI matrices for enhanced sample preparation, cleanup, and an increase in the ionization efficiencies of both native and permethylated glycans (Aim 4). Mass spectrometry based experiments can generate a tremendous amount of data that is cumbersome to analyze manually. There are numerous well-known proteomic software packages available but few that can comprehensively analyze glycomic datasets. We have developed MultiGlycan to analyze glycomic datasets and intend to expand its functionalities (Aim 5) to handle glycan isomers (Aim 1), multiplexed permethylated glycans (Aim 2), O-glycans (Aim 3), and glycans analyzed with MALDI-MS (Aim 4). The development of the proposed methods and algorithms will help us and collaborators to better understand the attributes and biomedical significance of glycan isomers in the development and progression of esophagus, breast, and liver cancer. We are also expecting the analytical tools and algorithms proposed here to be beneficial to other scientists who are interested in understanding the biological attributes of glycan isomers in other systems to benefit from.

糖组学已经成为生物学中一个有趣而又具有挑战性的研究领域。聚糖的功能在于 许多重要的生物学领域，例如但不限于：免疫系统、细胞发育、细胞免疫和细胞免疫。 在一些实施方案中，所述方法包括细胞分化/粘附、宿主-病原体相互作用、蛋白质信号传导和蛋白质稳定化。异常 糖基化与包括癌症、囊性纤维化和骨关节炎在内的几种疾病有关。 糖组学/糖蛋白质组学研究旨在量化和表征聚糖结构（包括连接和 位置异构体）、蛋白质附着位点和蛋白质的身份。大约50%的哺乳动物 蛋白质是糖基化的，但与非糖基化的蛋白质相比，它们的丰度相当低。此外，委员会认为， 许多聚糖可以占据蛋白质上相同的聚糖附着位点;即相同的蛋白质库可以 有几种不同类型的聚糖连接到同一位点，每一种具有潜在的不同功能或 特殊活动。蛋白聚糖根据其氨基酸连接位点分为两类： N-聚糖为天冬酰胺，O-聚糖为苏氨酸、丝氨酸和酪氨酸。 已经成功地用于研究细胞中的N-聚糖的策略是释放或分离N-聚糖。 用PNGase F酶从蛋白质中提取聚糖，并研究样品的总体聚糖组成。一 这种方法的缺点是，所谓的天然聚糖具有低电离效率，这使得它们的 质谱分析相当困难;然而，这种灵敏度问题可以通过全甲基化来克服。 聚糖聚糖具有许多异构体，如果存在不对称性，则难以通过LC-MS/MS对其进行准确分析。 异构体不能分离。该提议表明，我们能够分离全甲基化聚糖 在质谱分析之前，用加热的PGC柱对异构体进行分离（目的1），导致非常高的分离率。 灵敏的检测方法，可准确表征和定量生物样品中的聚糖异构体。虽然 以前曾报道过异构体聚糖的分离，以前的研究仅分离了天然和还原末端 标记的聚糖结构。由于全甲基化聚糖表现出至少两个电离效率， 比上述结构高几个数量级，灵敏度增加的重要性， 用于检测生理浓度下的结构，伴随着异构体分离， 不能夸大全甲基化聚糖（目标1）。 为了克服LC-MS样品之间电离效率的变化，我们成功地 具有各种稳定同位素组合的全甲基化聚糖， 来自细胞培养实验、生物流体和生物组织的样品之间的比较。 通过实施我们的多水平同位素标记策略（代谢15 N标记，18 O还原 末端标记和多重全甲基化），可以增加潜在多重样品的数量 生物液体和组织样本以及细胞培养的最大值从8个增加到16个和32个 样品，分别（目标2）。高通量异构体表征生物衍生的聚糖 可通过结合目标1和2中所述的方法获得样品。当使用PNGase F时， 广泛地从蛋白质中释放N-聚糖，对于O-聚糖，不存在或尚未发现这样的酶。 我们开发了一种快速的方法，RAIDR（快速氢氧化铵异丁酸O-聚糖 去糖基化反应），用于选择性释放O-聚糖; RAIDR留下蛋白质和N-聚糖 无损，这允许兼容的下游分析（目标3）。除了改进LC-MS分析， 方法，我们还建议将石墨烯纳米片和碳纳米颗粒添加到MALDI中 用于增强样品制备、净化和增加两种天然样品的电离效率的基质 和全甲基化聚糖（目标4）。基于质谱的实验可以产生大量的 手动分析起来很麻烦的数据。有许多著名的蛋白质组学软件包 可用，但很少有可以全面分析糖组学数据集。我们开发了MultiGlycan， 分析糖组学数据集，并打算扩展其功能（目标5）以处理聚糖异构体（目标1）， 多重全甲基化聚糖（目标2）、O-聚糖（目标3）和用MALDI-MS分析的聚糖（目标4）。 所提出的方法和算法的发展将有助于我们和合作者更好地理解 聚糖异构体在食管发育和进展中的属性和生物医学意义， 乳腺癌和肝癌。我们也期待这里提出的分析工具和算法是有益的 对了解其他系统中聚糖异构体的生物学属性感兴趣的其他科学家 从中受益